STRUCTURE-FUNCTION RELATIONSHIPS OF LYSOSOMAL ENZYMES

泰-萨克斯病是一种由突变引起的GM2神经节苷脂增多症 在HEXA基因中。我们已经破坏了小鼠的HEXA基因 同源基因打靶的胚胎干细胞株J-1 重组。两个靶向细胞系被用来产生嵌合体小鼠 将突变的等位基因传递给他们的后代。杂合子 小鼠被杂交产生纯合子的小鼠，以获得破坏的基因。 纯合子小鼠表现出完全缺乏β-氨基己糖苷酶 这是一种在泰-萨克斯病中缺失的酶。 β-己糖氨酸酶以两种主要同工酶A的形式存在，A是一种杂二聚体 以及B，B链的同源二聚体。每个亚单位 携带一种不同的活性，从而产生一种首选的光谱 被各自的同工酶降解的底物。例如，只有 杂二聚体能够降解神经节苷脂GM2。我们已经建造了 由α和β序列蛋白组成的嵌合亚单位 以确定负责底物偏好的区域。通过 这一策略我们已经确定了阿尔法亚单位底物 识别位点由不连续的氨基酸组成 来自N-末端和C-末端部分的序列 多肽。这项工作可能导致产生一种同源二聚体酶 可以降解CM2神经节苷脂，可能对酶有帮助 泰-萨克斯病的替代和/或基因治疗。

Tay-Sachs disease is a form of the GM2 gangliosidoses caused by mutations in the HEXA gene. We have disrupted the HEXA gene in the murine embryonic stem cell line, J-1, by gene targeting via homologous recombination. Two targeted cell lines were used to derive chimeric mice that transmitted the mutated allele to their offspring. Heterozygous mice were intercrossed to produce mice homozygous for the disrupted gene. The homozygous mice exhibit a complete deficiency of beta-hexosaminidase A, the enzyme that is absent in Tay-Sachs disease. Beta-Hexosaminidase exists as two predominant isozymes, A, a heterodimer of alpha and beta chains and B, a homodimer of B chains. Each subunit carries a different active giving rise to a preferred spectrum of substrates degraded by the respective isozyme. For example, only the heterodimer is able to degrade GM2 ganglioside. We have constructed chimeric subunits composed of proteins of alpha and beta sequences in order to identify the regions responsible for substrate preference. By this strategy we have determined that the alpha subunit substrate recognition site is composed of discontinuous stretches of amino acid sequence from both the N-terminal and C-terminal portions of the polypeptide. This work may lead to the creation of a homodimeric enzyme that can degrade CM2 ganglioside and which may be useful for enzyme replacement and/or gene therapy in Tay-Sachs disease.